Epidermodysplasia Verruciformis and Vδ2 γδ T-cell Expansion in STK4 Deficiency

The clinical penetrance of infectious diseases varies considerably among patients with inborn errors of immunity (IEI), even for identical genetic defects. This variability is influenced by pathogen exposure, healthcare access and host-environment interactions. We describe here a patient in his thirties who presented with epidermodysplasia verruciformis (EV) due to infection with a weakly virulent beta-papillomavirus (HPV38) and CD4+ T-cell lymphopenia. The patient was born to consanguineous parents living in the United States. Exome sequencing identified a previously unknown biallelic STK4 stop-gain mutation (p.Trp425X). The patient had no relevant history of infectious disease during childhood other than mild wart-like lesion on the skin, but he developed diffuse large B-cell lymphoma (DLBCL) and EBV viremia with a low viral load in his thirties. Despite his low CD4+ T-cell count, the patient had normal counts of CD3+ cells, predominantly double-negative T cells (67.4%), which turned out to be Vδ2+ γδ T cells. γδ T-cell expansion has frequently been observed in the 33 reported cases with STK4 deficiency. The Vδ2 γδ T cells of this STK4-deficient patient are mostly CD45RA-CD27+CCR7+ central memory γδT cells, and their ability to proliferate in response to T-cell activation was impaired, as was that of CD4+ T cells. In conclusion, γδ T-cell expansion may act as a compensatory mechanism to combat viral infection, providing immune protection in immunocompromised individuals. Supplementary Information The online version contains supplementary material available at 10.1007/s10875-024-01780-z.


Introduction
Epidermodysplasia verruciformis (EV) (OMIM 226400) is a rare autosomal recessive skin disorder characterized by persistent verrucae and pityriasis versicolor-like or warty cutaneous lesions due to impaired defenses against betapapillomaviruses (β-HPVs).This impairment increases the principal causal genes for EV, EVER1 and EVER2, by the late Gerard Orth led to the conclusion that patients with EV had an impaired keratinocyte-intrinsic immune response to β-HPVs [3].These two genetic disorders account for over 50% of cases of EV in patients with no particular susceptibility to viral or bacterial infections, a condition known as "typical EV".Further studies identified 24 EV patients homozygous for deleterious variants of the CIB1 gene, encoding the CIB1 protein of the CIB1-EVER1-EVER2 complex involved in the lifecycle of β-HPVs [4].Patients with typical EV develop skin lesions early in childhood, and 30-70% of EV patients subsequently develop squamous cell carcinoma, particularly those with HPV5 and HPV8 infections [5][6][7].By contrast, atypical EV is the condition identified in patients with other inborn errors of immunity (IEI) in addition to cutaneous β-HPV infection.Several patients with IEI, including AR RHOH, LCK, DOCK8 and STK4 deficiencies, have been reported to have histological findings consistent with EV due to β-HPVs [8].All these patients had CD4 + T-cell lymphopenia, but not all patients with T-cell deficiency develop EV; by contrast, penetrance is complete in patients with typical EV and AR CIB1-EVER1-EVER2 deficiency [1].
We report here a case in his thirties who presented at our immunology clinic with EV and T-cell lymphopenia.This patient was found to be homozygous for a previously unknown nonsense variant of STK4.Human STK4 (serine/ threonine kinase 4) deficiency was first described in 2012 as an autosomal recessive inborn error of immunity characterized by recurrent bacterial, viral and fungal infections due to profound CD4 + T lymphopenia in the first decade of life [9,10].STK4 is a highly conserved HIPPO pathway phosphorylated forkhead homeobox type O (FOXO) transcription factor controlling cell growth, tumorigenesis, and apoptosis.We characterized the clinical and immunological features of this patient diagnosed in his thirties with a condition typically identified early in life.

Clinical Study and Sequencing
Genomic DNA (gDNA) was isolated from the peripheral blood with the Quick-DNA™ Miniprep Plus Kit (ZYMO research, USA).Whole Exome Sequences (WES) were obtained at GENEWIZ with a Twist Human Comprehensive Exome kit and next-generation sequencing was performed on a NovaSeq XPlus sequencer.We followed the GATK best-practice workflow to establish our pipeline for variant calling [11].Briefly, raw-data quality metrics were calculated with FastQC and sequencing reads were mapped onto the reference genome (GRCh38) with BWA.PICARD and GATK4 were used to perform local realignment and basequality recalibration, respectively, to ensure high-quality base calls.The variant call format (VCF) file was annotated with VEP.Genetic diagnosis was facilitated by using a gene list for IEIs developed by the Expert Committee of the International Union of Immunological Societies (IUIS).β-HPVs were amplified from DNA extracted from the lesion swab, as previously described [12].Sanger sequencing was used to confirm the genotypes of β-HPVs and the STK4 variant.This study was approved by the institutional review boards of UTSW.

Statistical Analysis
All statistical analyses were performed in Prism 8.The statistical significance of quantitative differences between groups was assessed in unpaired t tests.P values below 0.05 were considered statistically significant.t-SNE map was used as a visualization tool for the qualitative assessment of cell population diversities for both the patient and a healthy control.The impact of limiting the markers used to construct a global t-SNE map to general lineage markers only was assessed by running t-SNE with the following markers alone: CD45RA, CD3, CD4, CD8, vδ2TCR, CD19, CD27, CD14, CD11c, CD56 and CD16.

Clinical Manifestations and Histological Characteristics of the Proband
The proband is a 31-year-old man living in Texas, the United States, who was born to consanguineous parents who had emigrated from the Middle East.He was referred to the immunology clinic for T-cell lymphopenia.He had no significant family history of immunodeficiency and first presented with flat warts on his hands at the age of three years.These warts subsequently spread diffusely over the scalp, ears, and upper extremities (Fig. 1A).The patient had normal growth and development, with no pneumonia, oral infections, or abscesses, and no significant history of prior infections other than pericarditis at the age of 12 years, and mild cellulitis not requiring drainage or antibiotics in his twenties.The proband is the only person from his family affected (Fig. 1B).At the age of 29 years, a mass causing sore throat and hoarseness was detected in the epiglottis.Biopsy and pathology analysis revealed a diffuse large B-cell lymphoma (DLBCL), with positive staining for CD20, CD79a, BCL2 and BLC6, but negative for CD10; 30% of the cells were positive for MIB-1 but MUM1 (IRF4) staining was negative, suggesting a germinal center-type lymphoma.The patient received four cycles of R-CHOP chemotherapy for stage IIa disease and complete remission was achieved.A PET-CT scan at two years of follow-up revealed an intensely hypermetabolic lung nodule in the left hilum.Biopsy results indicated an inflammatory process, suggesting a contained infection; no pathogens were identified.accounted for 89.1% of all DN T cells (Fig. 2B).CD4 + T lymphopenia and Vδ2 + T-cell expansion were not transient in the proband; these phenotypes were reproducible in blood tests performed a few months apart (Fig. 2C).Expansion of the γδ T-cell population is frequently reported in IEI [29], but expansion of the Vδ2 T-cell subset is a particularly interesting phenotype in the context of STK4 deficiency.

Investigating the Immunological Profile of Vδ2 T Cells in STK4 Deficiency
An expansion of γδ T cells has been observed in five patients with STK4 deficiency with or without EV [9,26,28].In humans, Vδ2 T cells are the predominant subset in the γδ T-cell population of peripheral blood; they respond to prenyl pyrophosphate metabolites (P-Ags) and have been implicated in antiviral and antitumor immunity [30].Most of the Vδ2 T cells from the proband were CD27 + CD45RA − , also exhibiting high CCR7 expression (Fig. S3), consistent with an innate-like Vγ9 + Vδ2 + central memory T-cell phenotype [31][32][33], but a minority had the CD27 lo CD45RA + effector phenotype (Fig. 3A).Vδ2 T cells from the proband displayed remarkably high levels of CD57 and CD56 expression (Fig. 3A), but did not express PD-1 or perforin (Fig. S2D).CD56 and CD57 are markers of exhausted γδ T cells.We therefore investigated the proliferative capacity of the cells.Proliferation rates in response to IL-2 with either PHA or CD3/CD28/CD2 stimulation were higher for CD4 + T cells than for Vδ2 + T cells.CD3/CD28/CD2 stimulation induced a more robust T-cell proliferation than PHA.The proband had low rates of CD4 + T-cell proliferation in response to both CD3/CD28/CD2 and PHA, but this impairment of proliferation was more marked for PHA stimulation (Fig. 3B), consistent with previous reports [9,16,18,24,[26][27][28].Vδ2 + T cells from the proband also displayed an impairment of proliferation relative to the healthy control.In conclusion, the cells of the expanded Vδ2 T-cell population from this STK4-deficient patient had mostly innate-like phenotypes and proliferated well in response to CD3/CD28/ CD2 stimulation, possibly as a compensatory mechanism for immune surveillance.

Discussion
Lymphopenia, flat warts, atopic dermatitis, EBV viremia and lymphoma are common presentations in inherited or acquired immunodeficiencies.In this case, CD4 + T-cell lymphopenia prompted evaluation at an adult immunology clinic, and genetic testing revealed STK4 deficiency.STK4 deficiency can cause combined immune deficiency, but this patient had only mild skin phenotypes and EBV viremia, Physical examination was unremarkable for the neck, lungs, heart, and abdomen.There was no lymphadenomegaly or hepatosplenomegaly.The patient had hypopigmented papules on his skin, flattened erythematous papules on the trunk that coalesced into plaques with a cobblestone pattern, and erythematous scaly plaques, mostly on the back of the hands and both wrists and behind the ears.He had scaly plaques on his scalp, upper arms, and forehead.Biopsy of the lesions on the back of the left wrist revealed acanthosis and papillomatosis, with numerous enlarged keratinocytes with a steel-gray cytoplasm, consistent with epidermodysplasia verruciformis (EV) (Fig. 1C).Amplification results for the DNA from the skin lesion swab revealed the presence of a β-HPV, which Sanger sequencing identified as HPV38, one of the β-HPVs known to cause EV [1] (Fig. S1).In addition, the EBV serology tests were positive for EBV-VCA and EBV-EA-IgG, together with EBV-DNA at 3.28 log IU/ml.EBV mRNA transcripts (EBNA2, BZLF1 and gp330/220) were undetectable in the RNA isolated from PBMCs (data now shown), suggesting a chronic latent EBV infection.

Characterization of the Genetic and Immunological Features
WES revealed that the proband was homozygous for a stopgain variant of the STK4 gene (NM_006282.5:c.1274G> A, p.Trp425X, Fig. S2A and S2B), which was confirmed by Sanger sequencing (Fig. 1D).Both his parents and one of his siblings were heterozygous for the variant.This variant is ultra-rare, absent from the gnomAD and "All of US" databases, predicted to be deleterious by SIFT, PolyPhen with a CADD score of 43, and classified as PVS1 [14].Laboratory tests showed the patient's hemoglobin levels and platelet counts to be normal, whereas he presented profound lymphopenia (total leukocyte count, 4.7 × 10 6 /L; lymphocytes 0.7 × 10 6 /L; neutrophils 3.3 × 10 6 /L).The patient had low CD4 + T-cell counts (136-292 cells/µl), normal B and NK cell proportions, a slightly high percentage of CD8 + T cells and a low CD4 + to CD8 + T-cell ratio (CD4/CD8 = 0.33-0.85;normal = 0.86-5).Immunoglobulin (Ig) profiling revealed low levels of IgM, with normal IgG and IgA levels (Table S2).
Interest in γδ T cells is growing due to their potent antiinfection and antitumor activities and their role in immunodeficiencies [29].An expansion of the γδ T-cell population has been reported in patients with atypical SCID, and has been shown to be associated with CMV infection and autoimmune cytopenia [29].Fourteen of the 33 STK4-deficient with no lymphoproliferative disorders or neutropenia.His immunoglobulin levels were normal, and he developed normal responses to vaccination against tetanus and diphtheria, indicating an absence of autoimmunity and no impairment of humoral immunity, consistent with the findings of normal B-cell levels in about two thirds of patients with STK4 deficiency (Table S3).The presence of DLBCL is unsurprising, as the relative risk of lymphoma is 8 to 10 times higher in patients with IEI than in immunocompetent individuals [34], with DLBCL diagnosed at earlier ages in IEI patients (41 vs. 55 years) [35].Including this case, 33 patients with STK4 deficiency have been reported in total, 10 of whom had lymphoma (Fig. 4).Another nine cases developed nonmalignant lymphoproliferative manifestations and five of

Fig. 1
Fig. 1 An STK4-deficient patient with HPV38-associated EV, atopic dermatitis and lymphoma.(A) Skin manifestations of scaly plaques and pigmentation on the forehead, and scaly erythematous plaques behind the ears, on the back of the hand and on the back of both wrists; (B) Family pedigree and variant segregation; (C) H&E staining of an

Fig. 2
Fig. 2 Immunophenotyping revealed low CD4 + T-cell levels, normal CD3 + T-cell levels and a high level of double-negative T cells in the proband.(A) t-SNE results for PBMCs, showing monocytes, NK, B, CD4 + and Vδ2 T cells; (B) T-cell subsets and expansion of the Vδ2